The U937 human pro monocytic cell line was obtained from European Collection of Cell Cultures. Non adherent U937 cells were cultured the in 10% FCS supplemented Inhibitors,Modulators,Libraries Roswell Park Memorial Institution 1640 with penicillin and strepto mycin at 37?C and 5% CO2. Cells were washed, counted using the trypan blue ex clusion assay and diluted to 1 106 cells ml in 10% FCS supplemented RPMI 1640. Cells were differentiated for 48 h with 32 nM phorbol 12 myristate 13 acetate at 37?C and 5% CO2. Cells were harvested and recounted using the trypan blue exclusion assay after differentiation and diluted to 1 106 cells ml in 2% FCS supplemented RPMI 1640. Into a 96 well plate was pipetted a 100 ul cell suspen sion and 80 ul 2% FCS supplemented medium. Plates were incubated at 37?C and 5% CO2 for 1 h or 24 h for non adherent or adherent cell lines, respectively.
Cytotoxicity of crude extracts and polyphenolic Inhibitors,Modulators,Libraries rich fractions Cytotoxicity was determined using the neutral red up take assay as described by Borenfreund et al. The final concentration of ethanol used in the cellular assays Inhibitors,Modulators,Libraries for the methanolic extract and polyphenolic rich fraction did not exceed 0. 1%. The cytotoxicity of crude extracts and polyphenolic rich samples was determined in pre seeded plates by addition of 20 ul medium, crude extracts or polyphenolic rich fractions and incubation for 72 h at 37?C and 5% CO2. Medium was replaced with 100 ul neutral red medium and incubated for 3 h after which plates were washed with PBS. Plates were left to dry, the dye dissolved using 100 ul neutral red eluent and the absorbance measured at 540 nm.
Attenuation of oxidative stress induced parameters in U937 cells The oxidant 2,2 azobis dihydro chloride is able to generate free radicals such as hydroxyls during thermolysis reactions. During generation of ROS, cells undergo cytotoxicity that can be detected as GSH depletion, Inhibitors,Modulators,Libraries apoptosis and lipid peroxida tion. These parameters can be measured using fluoromet ric and spectrophotometric assays. Induction of AAPH induced oxidative stress Into pre seeded U937 plates was pipetted 20 ul medium, Inhibitors,Modulators,Libraries positive control, crude extract, polyphenolic rich fraction or 10 mM Trolox and incubated for 1 h at 37 C and 5% CO2. In all experiments, except the ROS generation assays de scribed in Section Efficacy to protect against AAPH induced ROS generation, plates were washed with RPMI 1640, treated with 1. 5 mM AAPH and incubated for 48 h at 37?C and 5% CO2. All values were adjusted by subtraction of the blank. The results for percentage viability, apoptosis, lipid per oxidation and GSH depletion were expressed relative to the negative control using the following equation where, A intensity namely of triplicate negative con trol. A triplicate intensity of sample at a given concentration.